The present invention is in the field of pharmacogenomics, and is primarily directed to biallelic markers that are located in or in the vicinity of genes, which have an impact on arachidonic acid metabolism and the uses of these markers. The present invention encompasses methods of establishing associations between these markers and diseases involving arachidonic acid metabolism such as inflammatory diseases as well as associations between these markers and treatment response to drugs acting on arachidonic acid metabolism. The present invention also provides means to determine the genetic predisposition of individuals to such diseases and means to predict responses to such drugs.
The metabolites of arachidonic acid and related fatty acids, collectively termed eicosanoids, exhibit a wide range of biological activities affecting virtually every organ system in mammals. Eicosanoids are among the most important chemical mediators and modulators of the inflammatory reaction and contribute to a number of physiological and pathological processes (See Hardman J. G., Goodman, Gilman A., Limbird L. E.; Goodman and Gilman""s The Pharmacological Basis of Therapeutics, 9th edition, McGraw-Hill, N.Y., 1996).
Physiology, Pathophysiology and Pharmacological Importance of the Eicosanoids
The eicosanoids are extremely prevalent and have been detected in almost every tissue and body fluid. These lipids contribute to a number of physiological and pathological processes including inflammation, smooth muscle tone, hemostasis, thrombosis, parturition and gastrointestinal secretion. Once synthesized in response to a stimulus, the eicosanoids are not stored to any significant extent but are released immediately and act locally. After they act, they are quickly metabolized by local enzymes to inactive forms. Accordingly, the eicosanoids are categorized as autocrine agents or local hormones. They alter the activities of the cells in which they are synthesized and of adjoining cells. The nature of these effects may vary from one type of cell to another, in contrast with the more uniform actions of global hormones such as insulin, for example. Therefore, the eicosanoids, as local chemical messengers, exert a wide variety of effects in virtually every tissue and organ system.
The principal eicosanoids are the prostaglandins (PG), the thromboxanes (TX) and the leukotrienes (LT), though other derivatives of arachidonate, for example lipoxins, are also produced. They fall into different classes designated by letters and the main classes are further subdivided and designated by numbers.
Inflammatory and Immune Responses
Eicosanoids are lipid mediators of inflammation and play a central, often synergistic, role in numerous aspects of inflammatory responses and host defense. Prostaglandins and leukotrienes are released by a host of mechanical, thermal, chemical, bacterial, and other insults, and they contribute importantly to the genesis of the signs and symptoms of inflammation. The ability to mount an inflammatory response is essential for survival in the face of environmental pathogens and injury, although in some situations and diseases the inflammatory response may be exaggerated and sustained for no apparent beneficial reason. This is the case in numerous chronic inflammatory diseases and allergic inflammation. Acute allergic inflammation is characterized by increased blood flow, extravasation of plasma and recruitment of leukocytes. These events are triggered by locally released inflammatory mediators including eicosanoids and more particularly leukotrienes. The leukotrienes generally have powerful effects on vascular permeability and the leukotriene LTB4 is a potent chemoattractant for leukocytes and promotes exudation of plasma. The prostaglandins PGE2 and PGI2 markedly enhance edema formation and leukocyte infiltration in the inflamed region. Moreover, they potentiate the pain-producing activity of bradykinin.
The participation of arachidonic acid (AA) metabolism in inflammatory diseases such as rheumatoid arthritis, asthma and acute allergy is well established. Prostaglandins have been involved in inflammation, pain and fever. Pathological actions of leukotrienes are best understood in terms of their roles in immediate hypersensitivity and asthma. Lipoxygenases, e.g., 5-lipoxygenase (5-LO), 12-lipoxygenase (12-LO), 15-lipoxygenase A (15-LOA), and 15-lipoxygenase B (15-LOB), have been implicated in the pathogenesis of a variety of inflammatory conditions such as psoriasis and arthritis.
Cardiovascular System
The prostaglandins PGEs, PGF2 and PGD2 cause both vasodilation and vasoconstriction. Responses vary with concentration and vascular bed. Systemic blood pressure generally falls in response PGEs, and blood flow to most organs, including the heart, is increased. These effects are particularly striking in some hypertensive patients. Cardiac output is generally increased by prostaglandins of the E and F series. The importance of these vascular actions is emphasized by the participation of PGI2 and PGE2 in the hypotension associated with septic shock. The prostaglandins also have been implicated in the maintenance of patency of the ductus arteriosus. Thromboxane synthase (TXA2), also known as CYP5, is a potent vasoconstrictor. Leukotriene C4 synthase (LTC4) and the leokotriene LTD4 cause hypotension. The leukotrienes have prominent effects on the microvasculature. LTC4 and LTD4 appear to act on the endothelial lining of postcapillary venules to cause exudation of plasma; they are more potent than histamine in this regard. In higher concentrations, LTC4 and LTD4 constrict arterioles and reduce exudation of plasma.
Blood/Platelets
Prostanoids including prostaglandins and thromboxanes exhibit a wide variety of actions in various cells and tissues to maintain local homeostasis in the body. Eicosanoids modify the function of the formed elements of the blood. PGI2 controls the aggregation of platelets in vivo and contributes to the antithrombogenic properties of the intact vascular wall.
TXA2 is a major product of arachidonate metabolism in platelets and, as a powerful inducer of platelet aggregation and the platelet release reaction, is a physiological mediator of platelet aggregation. Pathways of platelet aggregation that are dependent on the generation of TXA2 are sensitive to the inhibitory action of aspirin, which inhibits the cyclooxygenase (COX) pathway. There has been considerable interest in the elucidation of the role played by prostaglandins and TXA2 in platelet aggregation and thrombosis and by PGI2 in the prevention of these events. The platelet thromboxane pathway is activated markedly in acute coronary artery syndromes and aspirin is beneficial in the secondary prevention of coronary and cerebrovascular diseases. PGI that is generated in the vessel wall may be the physiological antagonist of this system; it inhibits platelet aggregation and contributes to the nonthrombogenic properties of the endothelium. According to this concept, PGI2 and TXA2 represent biologically opposite poles of a mechanism for regulating platelet-vessel wall interaction and the formation of hemostatic plugs and intraarterial thrombi. There is interest in drugs which inhibit thromboxane synthase and modulate PGI2 production.
Smooth Muscle
Prostaglandins contract or relax many smooth muscles beside those of the vasculature. The leukotrienes contract most smooth muscles. In general, PGFs and PGD2 contract and PGEs relax bronchial and tracheal muscle. LTC4 and LTD4 are bronchoconstrictors. They act principally on smooth muscle in peripheral airways and are 1000 times more potent than histamine both in vitro and in vivo. They also stimulate bronchial mucus secretion and cause mucosal edema. A complex mixture of chemical messengers is released when sensitized lung tissue is challenged by the appropriate antigen. Various prostaglandins and leukotrienes are prominent components of this mixture. Response to the leukotrienes probably dominates during allergic constriction of the airway. Evidence for this conclusion is the ineffectiveness of inhibitors of cycloxygenase and of histaminergic antagonists in the treatment of human asthma and the protection afforded by leukotriene antagonists in antigen induced bronchoconstriction. A particularly important role for the cysteinyl-leukotrienes (LTC4, LTD4, and LTE4) has been suggested in pathogenesis of asthma, which is now recognized as a chronic inflammatory condition. They are potent spasmogens causing a contraction of bronchiolar muscle and an increase in mucus secretion.
Gastric and Intestinal Secretions
PGEs and PGI2 inhibit gastric acid secretion stimulated by feeding, histamine or gastrin. Mucus secretion in the stomach and small intestine is increased by PGEs. These effects help to maintain the integrity of the gastric mucosa and are referred to as the cytoprotectant properties of PGEs. Furthermore, PGEs and their analogs inhibit gastric damage caused by a variety of ulcerogenic agents and promote healing of duodenal and gastric ulcers. Cytoprotection is of therapeutic importance and PGE1 analogs are used for the prevention of gastric ulcers.
Kidney and Urine Formation
Prostaglandins modulate renal blood flow and may serve to regulate urine formation by both renovascular and tubular effects. Increased biosynthesis of prostaglandins has been associated with Bartter""s syndrome, a rare disease, characterized by urinary wasting of K+. Leukotrienes have been involved in the pathophysiology of glomerular immune injury.
Reproduction and Parturition
Much interest is attached to the possible involvement of prostaglandins in reproductive physiology. Lowered concentrations of prostaglandins in semen have been implicated in male infertility. Prostaglandins are also thought to contribute to the symptoms of primary dysmenorrhea. Inhibitors of cyclooxygenase are effective in relieving the symptoms of this condition. Elevated levels of prostaglandins are involved in onset of labor. Inhibitors of cyclooxygenase increase the length of gestation and interrupt premature labor.
Cancer Metastasis
Tumors in animals and certain spontaneous human tumors are accompanied by increased concentrations of local or circulating prostaglandins. Eicosanoids have been shown to be involved in various aspects of neoplasia including cell transformation, tumor promotion, tumor cell growth, and metastasis. Some studies have implicated platelet aggregation and the effects of prostaglandins and hydroxyeicosatetraenoic acid (12-HETE) in the hematogenous metastasis of tumors.
Many of the products of arachidonic acid metabolism are potent mediators of physiological responses and contribute to disorders of development, cellular function, tissue repair, and host defenses in a number of diseases.
Arachidonic Acid Metabolism and Biosynthesis of Eicosanoids
The primary source of eicosanoids in mammalian systems is the metabolic products of arachidonic acid. After stimulation by trauma, infection, or inflammation, translocated phospholipases, especially phospholipase A2, act on membrane phospholipids to liberate arachidonic acid. Once released, arachidonate is metabolized to oxygenated products by several distinct enzyme pathways, including cyclooxygenases, several lipoxygenases, and cytochrome P450s (CYP). The specific enzyme pathway involved determines, which products are formed.
Release of Arachidonic Acid from Cell Membranes and its Regulation
The eicosanoids are a family of substances produced from the polyunsaturated fatty acid arachidonic acid, which is present in plasma-membrane phospholipids. The first rate-limiting step in the biosynthesis of eicosanoids is the release of arachidonic acid from the membrane, a process that is mainly catalyzed by cytosolic phosholipase A2 (cPLA2). The synthesis of eicosanoids begins when a stimulus such as a hormone, a neurotransmitter, a drug or a toxic agent activates cytosolic phospholipase A2. This arachidonic acid specific phospholipase plays a major role in the cell signaling events that initiate the arachidonate cascade. One important trigger of arachidonate release and eicosanoid synthesis involves tissue injury and inflammation.
The activities of many enzymes are regulated by calmodulins (CAL) that serve as calcium sensors in eukaryotic cells. The binding of Ca2+ to multiple sites in calmodulin induces a major conformational change that converts it from an inactive to an active form. Activated calmodulin then binds to many enzymes and target proteins in the cell, modifying their activities and thereby regulating various metabolic pathways. Calmodulins are involved in a number of processes regulated by Ca2+ including smooth muscle contraction, neurotransmission, apoptosis, cell cycle progression and gene expression. Calmodulins also participate in the regulation of arachidonate release. They directly stimulate cytosolic phospholipase A2, whereas calmodulin antagonists inhibit enzyme activity and the release of arachidonic acid.
Annexins (ANX) are a family of multifunctional calcium and phospholipid-binding proteins, they belong to a family of proteins that interact with phospholipids in a Ca2+ dependant manner.
Annexins have been implicated in the pathogenesis of benign and malignant neoplasms of different origins. Moreover, several annexins have also been involved in autoimmune diseases such as systemic lupus erythematosus, rheumatoid arthritis and inflammatory bowl disease. Numerous physiological functions have been attributed to annexins including regulation of membrane traffic during exocytosis and endocytosis, mediation of cytoskeletal-membrane interactions, membrane receptor function, regulation of membrane-dependent enzymes, mitogenic signal transduction, transmembrane ion channel activity, cell-cell adhesion, antiinflammatory properties, inhibition of blood coagulation and inhibition of phospholipase A2. Annexins have been suggested as regulators of prostaglandin metabolism and of the arachidonate cascade as a result of their inhibitory effect on phospholipase A2. It is still a matter of debate as to whether inhibition of phospholipase A2 is the result of calcium-dependent sequestration of phospholipids (substrate depletion mechanism) or a direct effect of the annexins acting via protein-protein interactions. Calpactin I (light chain) is the cellular ligand of annexin II and induces its dimerization. Annexin II and calpactin I (CALPA) constitute a calcium binding complex composed of two light chains (calpactin I) and two heavy chains (annexin II). Calpactin I may function as regulator of annexin II phosphorylation.
The activities of phospholipase A2, annexins and calmodulins are common points of regulation in the formation of all eicosanoids.
Downstream of phospholipase A2, the varying eicosanoid-pathway enzymes found in particular cell types determine which eicosanoids are synthesized in response to particular stimuli.
Cyclooxygenase Pathway
This pathway initiated by cyclooxygenase (COX) leads ultimately to formation of the cyclic endoperoxides, prostaglandins (PG), and thromboxanes (TX). There are two isoforms of the cyclooxygenase, COX-1 and COX-2. The former is constitutively expressed in most cells. In contrast, COX-2 is not normally present but may be induced by certain factors such as cytokines and growth factors. The cyclooxygenases have two distinct activities: an endoperoxidase synthase activity that oxygenates and cyclizes the unesterified precursor fatty acid to form the cyclic endoperoxide PGG and a peroxidase activity that converts PGG to PGH. PGG and PGH are chemically unstable, but they can be transformed enzymatically into a variety of products, including PGI, TXA2, PGE, PGF or PGD. Isomerases lead to the synthesis of PGE2 and PGD2, whereas PGI2 is formed from PGH2 through prostacyclin synthase. TXA2 is formed by thromboxane synthase. Although most tissues are able to synthesize the PGG and PGH intermediates from free arachidonate, the fate of these precursors varies in each tissue and depends on the complement of enzymes that are present and on their relative abundance. For example, lung and spleen are able to synthesize the whole range of products. In contrast, platelets contain thromboxane synthase as the principal enzyme that metabolizes PGH, while endothelial cells contain primarily prostacyclin synthase.
Lipoxygenase Pathways
Lipoxygenases are a family of cytosolic enzymes that catalyze the oxygenation of fatty acids to corresponding lipid hydroperoxides. Arachidonate is metabolized to HPETE (hydroperoxyeicosatetraenoic acid), which is then converted either enzymatically or non-enzymatically to 12-HETE (hydroxyeicosatetraenoic acid). HPETEs may further be converted to hepoxilins and lipoxins. Lipoxygenases differ in their specificity for placing the hydroperoxy group, and tissues differ in the lipoxygenases they contain. These enzymes are referred to as 12-, 15-, 5- and 8-lipoxygenases according to the oxygenation sites in arachidonic acid as substrate.
The lipoxygenases catalyze reactions and generate products of potential relevance to membrane remodeling, cell differentiation and inflammation. Products of the 15-LO pathway could contribute to the pathophysiology of allergic airway inflammation while products of the 12-LO pathway have been implicated in cancer metastasis, psoriasis and inflammation.
Various biological activities have been reported for the 12-lipoxygenase metabolites of arachidonic acid. As other eicosanoids, they are important chemical mediators and modulators of the inflammatory reaction. 12-HETE is the major arachidonic acid metabolite of 12-lipoxygenase and seems to be implicated in a wide-spectrum of biological activities such as stimulation of insulin secretion by pancreatic tissue, suppression of renin production, chemoattraction of leukocytes and initiation of growth-related signaling events, such as activation of oncogenes, protein kinase C, and mitogen-activated protein kinases. 12-lipoxygenase activity and 12-HETE production are also important determining factors in tumor cell metastasis and have been implicated in human prostate cancer and breast cancer (Honn et al., Cancer Metastasis Rev., 13:365-396, 1994, Gao et al., Adv. Exp. Med. Biol., 407:41-53, 1997; Natarajan et al., J. Clin. Endocr. Metab., 82:1790-1789, 1997,). Further, 12-HETE has also been implicated in inflammatory skin diseases such as psoriasis (Hussai et al., Am. J. Physiol., 266:243-253, 1994). As mentioned above, metabolism of arachidonic acid by 12-lipoxygenase further generates lipoxins and hepoxillins. Lipoxins play the role of both immunologic and hemodynamic regulators and a variety of biological activities have been reported for hepoxillins which are related to the release of intracellular calcium and the opening of potassium channels (Yamamoto et al., Pro. Lipid Res., 36:23-41, 1997).
The 5-lipoxygenase (5-LO) is perhaps the most important of these enzymes since it leads to the synthesis of leukotrienes. Activation of the 5-LO enzyme involves its docking to a protein termed 5-lipoxygenase-activating protein (FLAP). This binding activates the enzyme, results in its association with the cell membrane and increased synthesis of 5-HPETE and leukotrienes. Leukotriene A (LTA) synthase is associated with 5-lipoxygenase and promotes the rearrangement of 5-HPETE to an unstable intermediate LTA4; which may be transformed to LTB4 by leukotriene A4 hydrolase (LTA4H); alternatively, it may be conjugated with glutathione by LTC4 synthase to form LTC4. LTA4 hydrolase is a pivotal element in leukotriene biosynthesis. Omega-oxidation is regarded as the major pathway for the catabolism of LTB4. This reaction is catalyzed by LTB4 omega-hydroxylase (LTB4H3) also called CYP4F2. LTD4 is produced by the removal of glutamic acid from LTC4 and LTE4 results from the subsequent cleavage of glycine; the reincorporation of glutamic acid yields LTF4.
Epoxygenase Pathway
Arachidonate is metabolized to a variety of metabolites by enzymes that contain cytochrome P450. The epoxygenase pathway of the arachidonic acid cascade leads to the formation of epoxyeicosatrienoic acids (EETs) and dihydroxyeicosatrienoic acids (DHETs). CYP2J2 is a human cytochrome P450 arachidonic acid epoxygenase expressed in extrahepatic tissues and particularly in the intestine. In addition to the known effects on intestinal vascular tone, CYP2J2 products may be involved in the release of intestinal neuropeptides, control of intestinal motility and modulation of intestinal fluid/electrolyte transport.
Eicosanoid Receptors
The diversity of the effects of eicosanoids is explained by the existence of a number of distinct receptors that mediate their actions. All prostaglandin receptors identified to date are coupled to effector mechanisms through G proteins. Distinct receptors for leukotrienes also have been identified in different tissues, all of these appear to activate phospholipase C.
Therapeutic Agents Interacting with Arachidonic Acid Metabolism
Because of their involvement in so many disease states, there has been a considerable effort to develop effective inhibitors to the formation or action of the eicosanoids. The drugs that influence the eicosanoid pathways are the most commonly used drugs in the world today. Their major uses are to reduce pain, fever and inflammation. Several classes of drugs, most notably the nonsteroidal antiinflammatory drugs (NSAIDs) owe their therapeutic effects to blockade of the formation of eicosanoids. Selective inhibitors of arachidonic acid metabolism also have an important therapeutic value. Inhibition of cyclooxygenase (COX), the enzyme responsible for the biosynthesis of the prostaglandins and certain related autacoids, generally is thought to be a major facet of the mechanism of NSAIDs. Aspirin and newer, widely used drugs belong to the NSAIDs. All NSAIDs are antipyretic, analgesic and antiinflammatory but there are important differences in their activities and in their side effects. The reasons for such differences are not fully understood. Side effects of these drugs include gastrointestinal ulceration, disturbances in platelet function, changes in renal function and hypersensitivity reactions. It is now appreciated that there are two forms of cyclooxygenase (COX), inhibition of COX-2 is thought to mediate the antipyretic, analgesic and antiinflammatory action of NSAIDs, whereas the simultaneous inhibition of COX-1 may result in unwanted side effects. Efforts are under way to identify COX-2 specific agents. But, it is also possible that enhanced generation of lipoxygenase products, due to the diversion of arachidonic acid metabolism from the cyclooxygenase pathway towards the lipoxygenase pathways, contributes to some of the side effects. Effort is being devoted to a search for drugs that will produce more selective interventions by acting farther along the biosynthetic pathways. Several compounds have been described that selectively antagonize responses to TXA2 and to PGH2. Some are receptor antagonists others directly inhibit thromboxane synthase.
Advances in understanding the pathobiology of the inflammatory process has suggested several novel approaches for development of drugs to block this process. These include phospholipase A2 inhibitors. Glucocorticoids are thought to have an effect on arachidonic acid metabolism through the induction of lipocortin that inhibits phospholipase A2.
NSAIDs generally do not inhibit the formation of other eicosanoids such as the lipoxygenase-produced leukotrienes. Substantial evidence indicates that leukotrienes contribute to the inflammatory response through a variety of effects. Leukotrienes have been implicated as mediators of inflammation and immediate hypersensitivity reactionsxe2x80x94in particular, human bronchial asthmaxe2x80x94and thus considerable effort has been done to develop either inhibitors of the production or blockers of the action of the actions of these mediators. Various therapeutic approaches have been used including 5-lipoxygenase inhibitors, which block leukotriene formation, or cysteinyl leukotriene receptor antagonists, which block receptor function. LTC4 synthase is another key step in biosynthesis of leukotrienes and represents another possible site for therapeutic intervention. Drugs targeting leukotriene biosynthesis are being tested and used for their utility in the treatment of various inflammatory conditions.
Most of these drugs are efficacious in providing relief but all available agents have associated, and sometimes severe, toxicity. Certain individuals display intolerance to aspirin and to other drugs acting on arachidonic acid metabolism; this is manifest by symptoms that range from liver toxicity, gastric and intestinal ulceration, disturbance in platelet function, renal injury, nephritis, vasomotor rhinitis with profuse watery secretions, angioneurotic edema, generalized urticaria, and bronchial asthma to laryngeal edema and bronchoconstriction, hypotension, and shock. The underlying mechanism for these severe side effects is not known. Moreover, while these agents have been highly useful for treatment of acute, self-limited inflammatory conditions; their ability to modify disease progression in chronic inflammatory settings remains an area of controversy. The complexity of the highly regulated pathways and enzymes that lead to the formation of the eicosanoids, has limited the precise identification of the metabolites and enzymes in the arachidonic acid cascade, which play the causal role in pathologies or in side effects to some drugs.
Pharmacogenomics and Arachidonic Acid Metabolism
The vast majority of common diseases, such as cancer, hypertension, diabetes and some inflammatory diseases are polygenic, meaning that they are caused by multiple genes. In addition, these diseases are modulated by environmental factors such as pollutants, chemicals and diet. This is why many diseases are called multifactorial; they result from a synergistic combination of factors, both genetic and environmental. Therapeutic management and drug development could be markedly improved by the identification of specific genetic polymorphisms that determine and predict patient susceptibility to diseases or patient responses to drugs.
To assess the origins of individual variations in disease susceptibility or drug response, pharmacogenomics uses the genomic technologies to identify polymorphisms within genes which are part of biological pathways involved in disease susceptibility, etiology, and development, or more specifically in drug response pathways responsible for a drug""s efficacy, tolerance or toxicity. It can provide tools to refine the design of drug development by decreasing the incidence of adverse events in drug tolerance studies, by better defining patient subpopulations of responders and non-responders in efficacy studies and, by combining the results obtained therefrom, to further allow better enlightened individualized drug usage based on efficacy/tolerance prognosis. Pharmacogenomics can also provide tools to identify new targets for designing drugs and to optimize the use of already existing drugs, in order to either increase their response rate and/or exclude non-responders from corresponding treatment, or decrease their undesirable side effects and/or exclude from corresponding treatment patients with marked susceptibility to undesirable side effects. However, for pharmacogenomics to become clinically useful on a large scale, molecular tools and diagnostics tests must become available.
Inflammatory reactions, which are involved in numerous diseases, are highly relevant to pharmacogenomics both because they are at the core of many widespread serious diseases, and because targeting inflammation pathways to design new efficient drugs includes numerous risks of potentiating serious side effects. Arachidonic acid metabolism is particularly relevant since its products, the eicosanoids, are powerful inflammatory molecules and play a role in a number of physiological functions.
Genetic Analysis of Complex Traits
Until recently, the identification of genes linked with detectable traits has relied mainly on a statistical approach called linkage analysis. Linkage analysis is based upon establishing a correlation between the transmission of genetic markers and that of a specific trait throughout generations within a family. Linkage analysis involves the study of families with multiple affected individuals and is useful in the detection of inherited-traits, which are caused by a single gene, or possibly a very small number of genes. Linkage analysis has been successfully applied to map simple genetic traits that show clear Mendelian inheritance patterns and which have a high penetrance (the probability that a person with a given genotype will exhibit a trait). About 100 pathological trait-causing genes have been discovered using linkage analysis over the last 10 years.
But, linkage studies have proven difficult when applied to complex genetic traits. Most traits of medical relevance do not follow simple Mendelian monogenic inheritance. However, complex diseases often aggregate in families, which suggests that there is a genetic component to be found. Such complex traits are often due to the combined action of multiple genes as well as environmental factors. Such complex trait, include susceptibilities to heart disease, hypertension, diabetes, cancer and inflammatory diseases. Drug efficacy, response and tolerance/toxicity can also be considered as multifactoral traits involving a genetic component in the same way as complex diseases. Linkage analysis cannot be applied to the study of such traits for which no large informative families are available. Moreover, because of their low penetrance, such complex traits do not segregate in a clear-cut Mendelian manner as they are passed from one generation to the next. Attempts to map such diseases have been plagued by inconclusive results, demonstrating the need for more sophisticated genetic tools.
Knowledge of genetic variation in the arachidonic acid cascade is important for understanding why some people are more susceptible to disease involving arachidonic acid metabolites or respond differently to treatments targeting arachidonic acid metabolism. Ways to identify genetic polymorphism and to analyze how they impact and predict disease susceptibility and response to treatment are needed.
Although the genes involved in arachidonic acid metabolism represent major drug targets and are of high relevance to pharmaceutical research, we still have scant knowledge concerning the extent and nature of sequence variation in these genes and their regulatory elements. For example, the cDNA and part of the genomic sequence for human 12-lipoxygenase have been cloned and sequenced (Izumi et al., Proc. Natl. Acad. Sci. USA, 87:7477-7481, 1990; Funk et al., Proc. Natl. Acad. Sci. USA, 87:5638-5642, 1990; Yoshimoto et al., Biochem. Biophys. Res. Commun., 172:1230-1235, 1990, Yoshimoto, et al., J. Biol. Chem., 267:24805-24809, 1992). However, the complete genomic sequence of the 12-lipoxygenase, including its regulatory elements, have not been described.
In the cases where polymorphisms have been identified, the relevance of the variation is rarely understood. While polymorphisms hold promise for use as genetic markers in determining which genes contribute to multigenic or quantitative traits, suitable markers and suitable methods for exploiting those markers have not been found and brought to bare on the genes related to arachidonic acid metabolism.
The present invention is based on the discovery of a set of novel eicosanoid-related biallelic markers. See Table 7(A-B). These markers are located in the coding regions as well as non-coding regions adjacent to genes which express proteins associated with arachidonic acid metabolism. The position of these markers and knowledge of the surrounding sequence has been used to design polynucleotide compositions which are useful in determining the identity of nucleotides at the marker position, as well as more complex association and haplotyping studies which are useful in determining the genetic basis for disease states involving arachidonic acid metabolism. In addition, the compositions and methods of the invention find use in the identification of the targets for the development of pharmaceutical agents and diagnostic methods, as well as the characterization of the differential efficacious responses to and side effects from pharmaceutical agents acting on arachidonic acid metabolism.
The present invention further stems from the isolation and characterization of the genomic sequence of the 12-lipoxygenase gene including its regulatory regions and of the complete cDNA sequence encoding the 12-lipoxygenase enzyme. Oligonucleotide probes and primers hybridizing specifically with a genomic sequence of 12-lipoxygenase are also part of the invention. Furthermore, an object of the invention consists of recombinant vectors comprising any of the nucleic acid sequences described in the present invention, and in particular of recombinant vectors comprising the promoter region of 12-lipoxygenase or a sequence encoding the 12-lipoxygenase enzyme, as well as cell hosts comprising said nucleic acid sequences or recombinant vectors. The invention also encompasses methods of screening of molecules which, modulate or inhibit the expression of the 12-lipoxygenase gene. The invention is also directed to biallelic markers that are located within the 12-lipoxygenase genomic sequence, these biallelic markers representing useful tools in order to identify a statistically significant association between specific alleles of 12-lipoxygenase gene and one or several disorders related to asthma and/or hepatotoxicity.
A first embodiment of the invention encompasses polynucleotides consisting of, consisting essentially of, or comprising a contiguous span of nucleotides of a sequence selected as an individual or in any combination from the group consisting of SEQ ID Nos. 1-70, 72-418, 425-489, 491-530, 532-539, and 541-652, and the complements thereof; preferably SEQ ID Nos. 651-652, 655-724, 726-1072, 1079-1143, 1145-1184, 1186-1193, and 1195-1304, and the complements thereof, or more preferably from SEQ ID Nos. 651-652, 680-724, 726-1072, 1079-1143, 1145-1184, 1186-1193, and 1195 -1300, and the complements thereof, wherein said contiguous span is at least 6, 8, 10, 12, 15, 20, 25, 30, 35, 40, 50, 75, 100, 200, 500, or 1000 nucleotides in length, to the extent that such a length is consistent with the lengths of the particular Sequence ID. The present invention also relates to polynucleotides hybridizing under stringent or intermediate conditions to a sequence selected as an individual or in any combination from the group consisting of SEQ ID Nos. 1-70, 72-418, 425-489, 491-530, 532-539, and 541-652, and the complements thereof, preferably SEQ ID Nos. 651-652, 655-724, 726-1072, 1079-1143, 1145-1184, 1186-1193, and 1195-1304, and the complements thereof; or more preferably from SEQ ID Nos. 651-652, 680-724, 726-1072, 1079-1143, 1145-1184, 1186-1193, and 1195-1300, and the complements thereof. In addition, the polynucleotides of the invention encompass polynucleotides with any further limitation described in this disclosure, or those following, specified alone or in any combination: Said contiguous span may optionally include the eicosanoid-related biallelic marker in said sequence; Optionally either the original or the alternative allele of Table 9 may be specified as being present at said eicosanoid-related biallelic marker; Optionally either the first or the second allele of Tables 8 or 10 may be specified as being present at said eicosanoid-related biallelic marker; Optionally, said polynucleotide may consists of, or consist essentially of a contiguous span which ranges in length from 8, 10, 12, 15, 18 or 20 to 25, 35, 40, 50, 60, 70, or 80 nucleotides, or be specified as being 12, 15, 18, 20, 25, 35, 40, or 50 nucleotides in length and including an eicosanoid-related biallelic marker of said sequence, and optionally the original allele of Table 9 is present at said biallelic marker; Optionally, said biallelic marker may be within 6, 5, 4, 3, 2, or 1 nucleotides of the center of said polynucleotide or at the center of said polynucleotide; Optionally, the 3xe2x80x2 end of said contiguous span may be present at the 3xe2x80x2 end of said polynucleotide; Optionally, biallelic marker may be present at the 3xe2x80x2 end of said polynucleotide; Optionally, the 3xe2x80x2 end of said polynucleotide may be located within or at least 2, 4, 6, 8, 10, 12, 15, 18, 20, 25, 50, 100, 250, 500, or 1000 nucleotides upstream of an eicosanoid-related biallelic marker in said sequence, to the extent that such a distance is consistent with the lengths of the particular Sequence ID; Optionally, the 3xe2x80x2 end of said polynucleotide may be located 1 nucleotide upstream of an eicosanoid-related biallelic marker in said sequence; and Optionally, said polynucleotide may further comprise a label.
A second embodiment of the invention encompasses any polynucleotide of the invention attached to a solid support. In addition, the polynucleotides of the invention which are attached to a solid support encompass polynucleotides with any further limitation described in this disclosure, or those following, specified alone or in any combination: Optionally, said polynucleotides may be specified as attached individually or in groups of at least 2, 5, 8, 10, 12, 15, 20, or 25 distinct polynucleotides of the inventions to a single solid support; Optionally, polynucleotides other than those of the invention may attached to the same solid support as polynucleotides of the invention; Optionally, when multiple polynucleotides are attached to a solid support they may be attached at random locations, or in an ordered array; Optionally, said ordered array may be addressable.
A third embodiment of the invention encompasses the use of any polynucleotide for, or any polynucleotide for use in, determining the identity of one or more nucleotides at an eicosanoid-related biallelic marker. In addition, the polynucleotides of the invention for use in determining the identity of one or more nucleotides at an eicosanoid-related biallelic marker encompass polynucleotides with any further limitation described in this disclosure, or those following, specified alone or in any combination. Optionally, said eicosanoid-related biallelic marker may be in a sequence selected individually or in any combination from the group consisting of SEQ ID Nos. 1-70, 72-418, 425-489, 491-530, 532-539, and 541-652, and the complements thereof; preferably SEQ ID Nos. 651-652, 655-724, 726-1072, 1079-1143, 1145-1184, 1186-1193, and 1195-1304, and the complements thereof; or more preferably from SEQ ID Nos. 651-652, 680-724, 726-1072, 1079-1143, 1145-1184, 1186-1193, and 1195-1300, and the complements thereof; Optionally, said polynucleotide may comprise a sequence disclosed in the present specification; Optionally, said polynucleotide may consist of, or consist essentially of any polynucleotide described in the present specification; Optionally, said determining may be performed in a hybridization assay, sequencing assay, microsequencing assay, or an enzyme-based mismatch detection assay; Optionally, said polynucleotide may be attached to a solid support, array, or addressable array; Optionally, said polynucleotide may be labeled.
A fourth embodiment of the invention encompasses the use of any polynucleotide for, or any polynucleotide for use in, amplifying a segment of nucleotides comprising an eicosanoid-related biallelic marker. In addition, the polynucleotides of the invention for use in amplifying a segment of nucleotides comprising an eicosanoid-related biallelic marker encompass polynucleotides with any further limitation described in this disclosure, or those following, specified alone or in any combination: Optionally, said eicosanoid-related biallelic marker may be in a sequence selected individually or in any combination from the group consisting of SEQ ID Nos. 1-70, 72-418, 425-489, 491-530, 532-539, and 541-652, and the complements thereof; preferably SEQ ID Nos. 651-652, 655-724, 726-1072, 1079-1143, 1145-1184, 1186-1193, and 1195-1304, and the complements thereof; or more preferably from SEQ ID Nos. 651-652, 680-724, 726-1072, 1079-1143, 1145-1184, 1186-1193, and 1195-1300, and the complements thereof; Optionally, said polynucleotide may comprise a sequence disclosed in the present specification; Optionally, said polynucleotide may consist of, or consist essentially of any polynucleotide described in the present specification; Optionally, said amplifying may be performed by a PCR or LCR. Optionally, said polynucleotide may be attached to a solid support, array, or addressable array. Optionally, said polynucleotide may be labeled.
A fifth embodiment of the invention encompasses methods of genotyping a biological sample comprising determining the identity of a nucleotide at an eicosanoid-related biallelic marker. In addition, the genotyping methods of the invention encompass methods with any further limitation described in this disclosure, or those following, specified alone or in any combination: Optionally, said eicosanoid-related biallelic marker may be in a sequence selected individually or in any combination from the group consisting of SEQ ID Nos. 1-70, 72-418, 425-489, 491-530, 532-539, and 541-652, and the complements thereof; preferably SEQ ID Nos. 651-652, 655-724, 726-1072, 1079-1143, 1145-1184, 1186-1193, and 1195-1304, and the complements thereof; or more preferably from SEQ ID Nos. 651-652, 680-724, 726-1072, 1079-1143, 1145-1184, 1186-1193, and 1195-1300, and the complements thereof, Optionally, said method further comprises determining the identity of a second nucleotide at said biallelic marker, wherein said first nucleotide and second nucleotide are not base paired (by Watson and Crick base pairing) to one another; Optionally, said biological sample is derived from a single individual or subject; Optionally, said method is performed in vitro; Optionally, said biallelic marker is determined for both copies of said biallelic marker present in said individual""s genome; Optionally, said biological sample is derived from multiple subjects or individuals; Optionally, said method further comprises amplifying a portion of said sequence comprising the biallelic marker prior to said determining step; Optionally, wherein said amplifying is performed by PCR, LCR, or replication of a recombinant vector comprising an origin of replication and said portion in a host cell; Optionally, wherein said determining is performed by a hybridization assay, sequencing assay, microsequencing assay, or an enzyme-based mismatch detection assay.
A sixth embodiment of the invention comprises methods of estimating the frequency of an allele in a population comprising genotyping individuals from said population for an eicosanoid-related biallelic marker and determining the proportional representation of said biallelic marker in said population. In addition, the methods of estimating the frequency of an allele in a population of the invention encompass methods with any further limitation described in this disclosure, or those following, specified alone or in any combination: Optionally, said eicosanoid-related biallelic marker may be in a sequence selected individually or in any combination from the group consisting of SEQ ID Nos. 1-70, 72-418, 425-489, 491-530, 532-539, and 541-652, and the complements thereof; preferably SEQ ID Nos. 651-652, 655-724, 726-1072, 1079-1143, 1145-1184, 1186-1193, and 1195-1304, and the complements thereof; or more preferably from SEQ ID Nos. 651-652, 680-724, 726-1072, 1079-1143, 1145-1184, 1186-1193, and 1195-1300, and the complements thereof; Optionally, determining the frequency of a biallelic marker allele in a population may be accomplished by determining the identity of the nucleotides for both copies of said biallelic marker present in the genome of each individual in said population and calculating the proportional representation of said nucleotide at said eicosanoid-related biallelic marker for the population; Optionally, determining the frequency of a biallelic marker allele in a population may be accomplished by performing a genotyping method on a pooled biological sample derived from a representative number of individuals, or each individual, in said population, and calculating the proportional amount of said nucleotide compared with the total.
A seventh embodiment of the invention comprises methods of detecting an association between an allele and a phenotype, comprising the steps of a) determining the frequency of at least one eicosanoid-related biallelic marker allele in a case population, b) determining the frequency of said eicosanoid-related biallelic marker allele in a control population and; c) determining whether a statistically significant association exists between said genotype and said phenotype. In addition, the methods of detecting an association between an allele and a phenotype of the invention encompass methods with any further limitation described in this disclosure, or those following, specified alone or in any combination: Optionally, said eicosanoid-related biallelic marker may be in a sequence selected individually or in any combination from the group consisting of SEQ ID Nos. 1-70, 72-418, 425-489, 491-530, 532-539, and 541-652, and the complements thereof; preferably SEQ ID Nos. 651-652, 655-724, 726-1072, 1079-1143, 1145-1184, 1186-1193, and 1195-1304, and the complements thereof; or more preferably from SEQ ID Nos. 651-652, 680-724, 726-1072, 1079-1143, 1145-1184, 1186-1193, and 1195-1300, and the complements thereof; Optionally, said control population may be a trait negative population, or a random population; Optionally, each of steps a) and b) is performed on a single pooled biological sample derived from each of said populations; Optionally, each of said steps a) and b) is performed on a single pooled biological sample derived from each of said populations; Optionally, each of said steps a) and b) is performed separately on biological samples derived from each individual in said populations; Optionally, said phenotype is a disease involving arachidonic acid metabolism, a response to an agent acting on arachidonic acid metabolism, or a side effects to an agent acting on arachidonic acid metabolism; Optionally, the identity of the nucleotides at the biallelic markers in everyone of the following sequences: SEQ ID Nos. 1-70, 72-418, 425-489, 491-530, 532-539, and 541-652; preferably SEQ ID Nos. 651-652, 655-724, 726-1072, 1079-1143, 1145-1184, 1186-1193, and 1195-1304; or more preferably from SEQ ID Nos. 651-652, 680-724, 726-1072, 1079-1143, 1145-1184, 1186-1193, and 1195-1300 is determined in steps a) and b).
An eighth embodiment of the present invention encompasses methods of estimating the frequency of a haplotype for a set of biallelic markers in a population, comprising the steps of: a) genotyping each individual in said population for at least one eicosanoid-related biallelic marker, b) genotyping each individual in said population for a second biallelic marker by determining the identity of the nucleotides at said second biallelic marker for both copies of said second biallelic marker present in the genome; and c) applying a haplotype determination method to the identities of the nucleotides determined in steps a) and b) to obtain an estimate of said frequency. In addition, the methods of estimating the frequency of a haplotype of the invention encompass methods with any further limitation described in this disclosure, or those following, specified alone or in any combination: Optionally said haplotype determination method is selected from the group consisting of asymmetric PCR amplification, double PCR amplification of specific alleles, the Clark method, or an expectation maximization algorithm; Optionally, said second biallelic marker is an eicosanoid-related biallelic marker in a sequence selected from the group consisting of the biallelic markers of SEQ ID Nos. 1-70, 72-418, 425-489, 491-530, 532-539, and 541-652, and the complements thereof; preferably SEQ ID Nos. 651-652, 655-724, 726-1072, 1079-1143, 1145-1184, 1186-1193, and 1195-1304, and the complements thereof; or more preferably from SEQ ID Nos. 651-652, 680-724, 726-1072, 1079-1143, 1145-1184, 1186-1193, and 1195-1300, and the complements thereof; Optionally, the identity of the nucleotides at the biallelic markers in everyone of the sequences: SEQ ID Nos. 1-70, 72-418, 425-489, 491-530, 532-539, and 541-652; preferably SEQ ID Nos. 651-652, 655-724, 726-1072, 1079-1143, 1145-1184, 1186-1193, and 1195-1304; or more preferably from SEQ ID Nos. 651-652, 680-724, 726-1072, 1079-1143, 1145-1184, 1186-1193, and 1195-1300 is determined in steps a) and b).
A ninth embodiment of the present invention encompasses methods of detecting an association between a haplotype and a phenotype, comprising the steps of: a) estimating the frequency of at least one haplotype in a case population according to a method of estimating the frequency of a haplotype of the invention; b) estimating the frequency of said haplotype in a control population according to the method of estimating the frequency of a haplotype of the invention; and c) determining whether a statistically significant association exists between said haplotype and said phenotype. In addition, the methods of detecting an association between a haplotype and a phenotype of the invention encompass methods with any further limitation described in this disclosure, or those following, specified alone or in any combination: Optionally, said eicosanoid-related biallelic marker may be in a sequence selected individually or in any combination from the group consisting of SEQ ID Nos. 1-70, 72-418, 425-489, 491-530, 532-539, and 541-652, and the complements thereof; preferably SEQ ID Nos. 651-652, 655-724, 726-1072, 1079-1143, 1145-1184, 1186-1193, and 1195-1304, and the complements thereof; or more preferably from SEQ ID Nos. 651-652, 680-724, 726-1072, 1079-1143, 1145-1184, 1186-1193, and 1195-1300, and the complements thereof; Optionally, said control population may be a trait negative population, or a random population; Optionally, said phenotype is a disease involving arachidonic acid metabolism, a response to an agent acting on arachidonic acid metabolism, or a side effects to an agent acting on arachidonic acid metabolism; Optionally, the identity of the nucleotides at the biallelic markers in everyone of the following sequences: SEQ ID Nos. 1-70, 72-418, 425-489, 491-530, 532-539, and 541-652; preferably SEQ ID Nos. 651-652, 655-724, 726-1072, 1079-1143, 1145-1184, 1186-1193, and 1195-1304; or more preferably from SEQ ID Nos. 651-652, 680-724, 726-1072, 1079-1143, 1145-1184, 1186-1193, and 1195-1300 is included in the estimating steps a) and b).
A tenth embodiment of the present invention is a method of administering a drug or a treatment comprising the steps of: a) obtaining a nucleic acid sample from an individual; b) determining the identity of the polymorphic base of at least one eicosanoid-related biallelic marker or 12-LO-related biallelic marker according to the methods taught herein which is associated with a positive response to said drug or treatment, or at least one eicosanoid-related marker or 12-LO-related biallelic marker or which is associated with a negative response to said drug or treatment; and c) administering said drug or treatment to said individual if said nucleic acid sample contains at least one biallelic marker associated with a positive response to said drug or treatment, or if said nucleic acid sample lacks at least one biallelic marker associated with a negative response to said drug or treatment. In addition, the methods of the present invention for administering a drug or a treatment encompass methods with any further limitation described in this disclosure, or those following, specified alone or in any combination: optionally, said eicosanoid-related biallelic marker or 12-LO-related biallelic marker may be in a sequence selected individually or in any combination from the group consisting of SEQ ID Nos. 1-70, 72-418, 425-489, 491-530, 532-539, and 541-652, and the complements thereof; preferably SEQ ID Nos. 651-652, 655-724, 726-1072, 1079-1143, 1145-1184, 1186-1193, and 1195-1304, and the complements thereof; or more preferably from SEQ ID Nos. 651-652, 680-724, 726-1072, 1079-1143, 1145-1184, 1186-1193, and 1195-1300, and the complements thereof; or optionally, the administering step comprises administering the drug or the treatment to the individual if the nucleic acid sample contains said biallelic marker associated with a positive response to the treatment or the drug and the nucleic acid sample lacks said biallelic marker associated with a negative response to the treatment or the drug.
An eleventh embodiment of the present invention is a method of selecting an individual for inclusion in a clinical trial of a treatment or drug comprising the steps of: a) obtaining a nucleic acid sample from an individual; b) determining the identity of the polymorphic base of at least one eicosanoid-related biallelic marker or 12-LO-related biallelic marker which is associated with a positive response to the treatment or the drug, or at least one eicosanoid-related biallelic marker or 12-LO-related biallelic marker which is associated with a negative response to the treatment or the drug in the nucleic acid sample, and c) including the individual in the clinical trial if the nucleic acid sample contains said eicosanoid-related biallelic marker or 12-LO-related biallelic marker associated with a positive response to the treatment or the drug or if the nucleic acid sample lacks said biallelic marker associated with a negative response to the treatment or the drug. In addition, the methods of the present invention for selecting an individual for inclusion in a clinical trial of a treatment or drug encompass methods with any further limitation described in this disclosure, or those following, specified alone or in any combination: Optionally, said eicosanoid-related biallelic marker or 12-LO-related biallelic marker may be in a sequence selected individually or in any combination from the group consisting of SEQ ID Nos. 1-70, 72-418, 425-489, 491-530, 532-539, and 541-652, and the complements thereof; preferably SEQ ID Nos. 651-652, 655-724, 726-1072, 1079-1143, 1145-1184, 1186-1193, and 1195-1304, and the complements thereof, or more preferably from SEQ ID Nos. 651-652, 680-724, 726-1072, 1079-1143, 1145-1184, 1186-1193, and 1195-1300, and the complements thereof.
Additional embodiments are set forth in the Detailed Description of the Invention and in the Examples.
Table 1 contains the first five markers listed in the sequence listing and their corresponding SEQ ID numbers.
Tables 2A-C are a list of 12-LO-related biallelic markers.
Table 3 is a listing of currently available forensic testing systems and their characteristics as compared to the method of the invention.
Table 4 sets forth the number of biallelic markers (VNTRs) needed to obtain, in mean, a ratio of at least 106 or 108.
Table 5 provides an indication of the descriminatory potential of the systems of the invention.
Table 6 is a listing of probabilities for several different types of relationships and likelihood ratios.
Table 7A is a chart containing a list of all of the eicosanoid-related biallelic markers for each gene with an indication of the gene for which the marker is in closest physical proximity, an indication of whether the markers have been validated by microsequencing (with a Y indicating that the markers have been validated by microsequencing and an N indicating that it has not), and an indication of the identity and frequency of the least common allele determined by genotyping (with a blank left to indicate that the frequency has not yet been reported for some markers). The frequencies were determined from DNA samples collected from a random US Caucasian population. When the marker was determined to be homozygous at the particular location for the random US Caucasian population, the homozygous bases were recorded in the xe2x80x9cGenotyping Least Common Allele Frequencyxe2x80x9d column of Table 7A. For example, Seq. ID No. 16 was determined to be homozygous G/G at the biallelic marker position 478 in the US control population, therefore G/G was recorded in the xe2x80x9cGenotyping Least Common Allele Frequencyxe2x80x9d column.
Table 7B contains all of the eicosanoid-related biallelic markers provided in Table 7A; however, they are provided in shorter, easier to search sequences of 47 nucleotides. Accordingly, Table 7A begins with SEQ ID No. 1 and ends with SEQ ID No. 654, while Table 7B begins with SEQ ID No. 655 and ends with SEQ ID No. 1604 (SEQ ID Nos. 651-654 correspond to the genomic and protein sequences of the invention and are not repeated in Table 7B). Table 1 contains the first five markers listed in the sequence listing and their corresponding SEQ ID numbers in Tables 7A and 7B to illustrate the relationship between Tables 7A and 7B:
Table 7B is the same as Table 7A in that it is a list of all of the eicosanoid-related biallelic markers for each gene with an indication of the gene for which the marker is in closest physical proximity, an indication of whether the markers have been validated by microsequencing (with a Y indicating that the markers have been validated by microsequencing and an N indicating that it has not), and an indication of the identity and frequency of the least common allele determined by genotyping (with a blank left to indicate that the frequency has not yet been reported for some markers). However, the xe2x80x9cBiallelic Marker Position in SEQ ID No.xe2x80x9d for all of the eicosanoid-related biallelic markers provided in Table 7B is position 24 (representing the midpoint of the 47 mers that make up Table 7B). The frequencies were determined from DNA samples collected from a random US Caucasian population. When the marker was determined to be homozygous at the particular location for the random US Caucasian population, the homozygous bases were recorded in the xe2x80x9cGenotyping Least Common Allele Frequencyxe2x80x9d column of Table 7B. For example, Seq. ID No. 670 was determined to be homozygous G/G at the biallelic marker position 24 in the US control population, therefore G/G was recorded in the xe2x80x9cGenotyping Least Common Allele Frequencyxe2x80x9d column.
Tables 8, 9, and 10 are charts containing lists of the eicosanoid-related biallelic markers. Each marker is described by indicating its SEQ ID, the biallelic marker ID, and the two most common alleles. Table 8 is a chart containing a list of biallelic markers surrounded by preferred sequences. In the column labeled, xe2x80x9cPOSITION RANGE OF PREFERRED SEQUENCExe2x80x9d of Table 8 regions of particularly preferred sequences are listed for each SEQ ID, which contain an eicosanoid-related biallelic marker, as well as particularly preferred regions of sequences that do not contain an eicosanoid-related biallelic marker but, which are in sufficiently close proximity to an eicosanoid-related biallelic marker to be useful as amplification or sequencing primers.
Table 11 is a chart listing particular sequences that are useful for designing some of the primers and probes of the invention. Each sequence is described by indicating its Sequence ID and the positions of the first and last nucleotides (position range) of the particular sequence in the Sequence ID.
Table 12 is a chart listing microsequencing primers which have been used to genotype eicosanoid-related biallelic markers (indicated by an *) and other preferred microsequencing primers for use in genotyping eicosanoid-related biallelic markers. Each of the primers which falls within the strand of nucleotides included in the Sequence Listing are described by indicating their Sequence ID number and the positions of the first and last nucleotides (position range) of the primers in the Sequence ID. Since the sequences in the Sequence Listing are single stranded and half the possible microsequencing primers are composed of nucleotide sequences from the complementary strand, the primers that are composed of nucleotides in the complementary strand are described by indicating their SEQ ID numbers and the positions of the first and last nucleotides to which they are complementary (complementary position range) in the Sequence ID.
Table 13 is a chart listing amplification primers which have been used to amplify polynucleotides containing one or more eicosanoid-related biallelic markers. Each of the primers which falls within the strand of nucleotides included in the Sequence Listing are described by indicating their Sequence ID number and the positions of the first and last nucleotides (position range) of the primers in the Sequence ID. Since the sequences in the Sequence Listing are single stranded and half the possible amplification primers are composed of nucleotide sequences from the complementary strand, the primers that are composed of nucleotides in the complementary strand are defined by the SEQ ID numbers and the positions of the first and last nucleotides to which they are complementary (complementary position range) in the Sequence ID.
Table 14 is a chart listing preferred probes useful in genotyping eicosanoid-related biallelic markers by hybridization assays. The probes are 25-mers with an eicosanoid-related biallelic marker in the center position, and described by indicating their Sequence ID number and the positions of the first and last nucleotides (position range) of the probes in the Sequence ID. The probes complementary to the sequences in each position range in each Sequence ID are also understood to be a part of this preferred list even though they are not specified separately.
Table 15 is a table showing the results of the association study between biallelic marker haplotypes from the FLAP gene and asthma.
Table 16 is a table showing the results of the permutation test confirming the statistical significance of the association between asthma and biallelic marker haplotypes from the FLAP gene.
Table 17 is a table showing the results of the association study between 12 biallelic marker haplotypes from the 12-LO gene and asthma.
Table 18A is a table showing the results of allele frequency analysis between seventeen 12-LO biallelic markers and asthma.
Table 18B is a table showing the results of the association study between seventeen 12-LO biallelic marker haplotypes from the 12-LO gene and asthma.
Table 19 is a table showing the results of the association study between 12 biallelic marker haplotypes from the 12-LO gene and hepatotoxicity upon treatment with zileuton.
Table 20A is a table showing the results of the allele frequency analysis between seventeen 12-LO biallelic markers and hepatotoxicity upon treatment with zileuton.
Table 20B is a table showing the results of the association study between seventeen 12-LO biallelic marker haplotypes from the 12-LO gene and hepatotoxicity upon treatment with zileuton.
Table 21 is a table showing a summary of the association study results, permutation tests confirming the statistical significance of the association between asthma and biallelic marker haplotypes from the 12-LO gene, and permutation tests confirming the statistical significance of the association between secondary effects upon treatment with zileuton and biallelic marker haplotypes from the 12-LO gene.
Table 22 is a table showing a summary of the association study results, permutation tests confirming the statistical significance of the association between asthma and additional biallelic marker haplotypes from the 12-LO gene, and permutation tests confirming the statistical significance of the association between secondary effects upon treatment with zileuton and biallelic marker haplotypes from the 12-LO gene.
Table 23 is a chart containing a list of preferred 12-LO-related biallelic markers with an indication of the frequency of the least common allele determined by genotyping. Frequencies were determined in a random US Caucasian population, in an asthmatic population showing no side effects upon treatment with Zyflo(trademark) (ALTxe2x88x92) and in an asthmatic population showing elevated alanine aminotransferase levels upon treatment with Zyflo(trademark) (ALT+).